Discovery of pyrrolo[2,1-f][1,2,4]triazine-based inhibitors of adaptor protein 2-associated kinase 1 for the treatment of pain.

Adaptor protein 2-associated kinase 1 (AAK1) is a member of the Ark1/Prk1 family of serine/threonine kinases and plays a role in modulating receptor endocytosis. AAK1 was identified as a potential therapeutic target for the treatment of neuropathic pain when it was shown that AAK1 knock out (KO) mice had a normal response to the acute pain phase of the mouse formalin model, but a reduced response to the persistent pain phase. Herein we report our early work investigating a series of pyrrolo[2,1-f][1,2,4]triazines as part of our efforts to recapitulate this KO phenotype with a potent, small molecule inhibitor of AAK1. The synthesis, structure-activity relationships (SAR), and in vivo evaluation of these AAK1 inhibitors is described.

An amide-based sulfenamide prodrug of gamma secretase inhibitor BMS-708163 delivers parent drug from an oral conventional solid dosage form in male beagle dog.

The objective of this Letter is to report the first (to our knowledge) in vivo proof of concept for a sulfenamide prodrug to orally deliver a poorly soluble drug containing a weakly-acidic NH-acid from a conventional solid dosage formulation. This proof of concept was established using BMS-708163 (1), a gamma secretase inhibitor containing a weakly acidic primary amide NH-acid as the chemical handle for attaching a series of thiol-based promoieties via a sulfenamide linkage. Aqueous stabilities and solubilities are reported for a series of six sulfenamide prodrugs (2-7) of 1. The sulfenamide prodrug containing the cysteine methyl ester promoiety (5) was chosen for a orally-dosed PK study in male beagle dog comparing a solubilized formulation of 1 against a solid dosage form of 5 in a cross-over fashion at an equivalent molar dose of 3 mg/kg. Prodrug 5 delivered essentially a superimposable PK profile of 1 compared to the solubilized formulation of 1, without any detectable exposure of 5 in systemic circulation.

Linking (Pyr)1apelin-13 pharmacokinetics to efficacy: Stabilization and measurement of a high clearance peptide in rodents.

Apelin, the endogenous ligand for the APJ receptor, has generated interest due to its beneficial effects on the cardiovascular system. Synthesized as a 77 amino acid preproprotein, apelin is post-translationally cleaved to a series of shorter peptides. Though (Pyr)1apelin-13 represents the major circulating form in plasma, it is highly susceptible to proteolytic degradation and has an extremely short half-life, making it challenging to quantify. Literature reports of apelin levels in rodents have historically been determined with commercial ELISA kits which suffer from a lack of selectivity, recognizing a range of active and inactive isoforms of apelin peptide. (Pyr)1apelin-13 has demonstrated beneficial hemodynamic effects in humans, and we wished to evaluate if similar effects could be measured in pre-clinical models. Despite development of a highly selective LC/MS/MS method, in rodent studies where (Pyr)1apelin-13 was administered exogenously the peptide was not detectable until a detailed stabilization protocol was implemented during blood collection. Further, the inherent high clearance of (Pyr)1apelin-13 required an extended release delivery system to enable chronic dosing. The ability to deliver sustained doses and stabilize (Pyr)1apelin-13 in plasma allowed us to demonstrate for the first time the link between systemic concentration of apelin and its pharmacological effects in animal models.

The γ-Secretase Modulator, BMS-932481, Modulates Aß Peptides in the Plasma and Cerebrospinal Fluid of Healthy Volunteers.

The pharmacokinetics, pharmacodynamics, safety, and tolerability of BMS-932481, a γ-secretase modulator (GSM), were tested in healthy young and elderly volunteers after single and multiple doses. BMS-932481 was orally absorbed, showed dose proportionality after a single dose administration, and had approximately 3-fold accumulation after multiple dosing. High-fat/caloric meals doubled the Cmax and area under the curve and prolonged Tmax by 1.5 hours. Consistent with the preclinical pharmacology of GSMs, BMS-932481 decreased cerebrospinal fluid (CSF) Aß39, Aß40, and Aß42 while increasing Aß37 and Aß38, thereby providing evidence of γ-secretase enzyme modulation rather than inhibition. In plasma, reductions in Aß40 and Aß42 were observed with no change in total Aß; in CSF, modest decreases in total Aß were observed at higher dose levels. Increases in liver enzymes were observed at exposures associated with greater than 70% CSF Aß42 lowering after multiple dosing. Although further development was halted due to an insufficient safety margin to test the hypothesis for efficacy of Aß lowering in Alzheimer's disease, this study demonstrates that γ-secretase modulation is achievable in healthy human volunteers and supports further efforts to discover well tolerated GSMs for testing in Alzheimer's disease and other indications.

Inhibition of AAK1 Kinase as a Novel Therapeutic Approach to Treat Neuropathic Pain.

To identify novel targets for neuropathic pain, 3097 mouse knockout lines were tested in acute and persistent pain behavior assays. One of the lines from this screen, which contained a null allele of the adapter protein-2 associated kinase 1 (AAK1) gene, had a normal response in acute pain assays (hot plate, phase I formalin), but a markedly reduced response to persistent pain in phase II formalin. AAK1 knockout mice also failed to develop tactile allodynia following the Chung procedure of spinal nerve ligation (SNL). Based on these findings, potent, small-molecule inhibitors of AAK1 were identified. Studies in mice showed that one such inhibitor, LP-935509, caused a reduced pain response in phase II formalin and reversed fully established pain behavior following the SNL procedure. Further studies showed that the inhibitor also reduced evoked pain responses in the rat chronic constriction injury (CCI) model and the rat streptozotocin model of diabetic peripheral neuropathy. Using a nonbrain-penetrant AAK1 inhibitor and local administration of an AAK1 inhibitor, the relevant pool of AAK1 for antineuropathic action was found to be in the spinal cord. Consistent with these results, AAK1 inhibitors dose-dependently reduced the increased spontaneous neural activity in the spinal cord caused by CCI and blocked the development of windup induced by repeated electrical stimulation of the paw. The mechanism of AAK1 antinociception was further investigated with inhibitors of α2 adrenergic and opioid receptors. These studies showed that α2 adrenergic receptor inhibitors, but not opioid receptor inhibitors, not only prevented AAK1 inhibitor antineuropathic action in behavioral assays, but also blocked the AAK1 inhibitor-induced reduction in spinal neural activity in the rat CCI model. Hence, AAK1 inhibitors are a novel therapeutic approach to neuropathic pain with activity in animal models that is mechanistically linked (behaviorally and electrophysiologically) to α2 adrenergic signaling, a pathway known to be antinociceptive in humans.

Synthesis and evaluation of carbamate and aryl ether substituted pyrazinones as corticotropin releasing factor-1 (CRF1) receptor antagonists.

A series of pyrazinone-based compounds incorporating either carbamate or aryl ether groups was synthesized and evaluated as corticotropin-releasing factor-1 (CRF1) receptor antagonists. Structure-activity relationship studies led to the identification of highly potent CRF1 receptor antagonists 14a (IC50=0.74 nM) and 14b (IC50=1.9 nM). The synthesis, structure-activity relationships and in vitro metabolic stability properties of compounds in this series will be described.

Design and optimization of tricyclic gamma-secretase modulators.

Beginning with a diaminotriazine screening hit, several series of novel, tricyclic gamma-secretase modulators (GSMs) were designed. The SAR of several related series of GSMs is presented, and the in vivo profile of a lead molecule from the series is described.

Macrocyclic prolinyl acyl guanidines as inhibitors of ß-secretase (BACE).

The synthesis, evaluation, and structure-activity relationships of a class of acyl guanidines which inhibit the BACE-1 enzyme are presented. The prolinyl acyl guanidine chemotype (7c), unlike compounds of the parent isothiazole chemotype (1), yielded compounds with good agreement between their enzymatic and cellular potency as well as a reduced susceptibility to P-gp efflux. Further improvements in potency and P-gp ratio were realized via a macrocyclization strategy. The in vivo profile in wild-type mice and P-gp effects for the macrocyclic analog 21c is presented.

D-α-tocopheryl polyethylene glycol 1000 succinate-containing vehicles provide no detectable chemoprotection from oxidative damage.

The objective of this study was to evaluate potential protective effects of vehicles containing d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), which may impact nonclinical safety assessments of oxidative processes. This was achieved by evaluating plasma, liver and adrenal gland concentrations of d-α-tocopheryl succinate (TS) and d-α-tocopherol as well as oxidative status of plasma following oral dosing of TPGS-containing vehicles, intraperitoneal (IP) dosing of TS or ex vivo treatment of blood with H2O2. Male and female rats were dosed orally with formulations containing 5% or 40% TPGS (70 or 550 mg kg(-1) day(-1) TS, respectively) for 1 week. A control group was dosed orally with polyethylene glycol-400 (PEG-400; no vitamin E) and positive control animals received a single 100 mg kg(-1) day(-1) IP injection of TS. Whole blood from untreated animals was treated ex vivo with 5 or 50 mm H(2)O(2), with or without TS (0.5, 5, 50 or 500 µm) or ascorbate (1 mm), for 1 h. Oral TPGS treatments did not affect d-α-tocopherol concentrations in plasma or adrenal glands and caused only transient increases in liver. Concentrations of TS in plasma, liver and adrenal glands were undetectable in control animals, but increased in all other groups. Oral administration of TPGS did not reduce plasma lipid peroxidation in vivo. Substantially greater TS concentrations used ex vivo (100× greater than in vivo) were also unable to reduce lipid peroxidation in H2O2 -treated whole blood. These results provide evidence that administration of oral TPGS vehicles is unlikely to impact nonclinical safety assessments of pharmaceuticals.

Structure activity relationship studies of 3-arylsulfonyl-pyrido[1,2-a]pyrimidin-4-imines as potent 5-HT6 antagonists.

Comprehensive structure activity relationship (SAR) studies were conducted on a focused screening hit, 2-(methylthio)-3-(phenylsulfonyl)-4H-pyrido[1,2-a]pyrimidin-4-imine (1, IC50: 4.0 nM), as 5-HT6 selective antagonists. Activity was improved some 2-4 fold when small, electron-donating groups were added to the central core as observed in 19, 20 and 26. Molecular docking of key compounds in a homology model of the human 5-HT6 receptor was used to rationalize our structure-activity relationship (SAR) findings. In pharmacokinetic experiments, compound 1 displayed good brain uptake in rats following intra-peritoneal administration, but limited oral bioavailability.

Discovery of BMS-986308: A Renal Outer Medullary Potassium Channel Inhibitor for the Treatment of Heart Failure.

Recent literature reports highlight the importance of the renal outer medullary potassium (ROMK) channel in renal sodium and potassium homeostasis and emphasize the potential impact that ROMK inhibitors could have as a novel mechanism diuretic in heart failure patients. A series of piperazine-based ROMK inhibitors were designed and optimized to achieve excellent ROMK potency, hERG selectivity, and ADME properties, which led to the identification of compound 28 (BMS-986308). BMS-986308 demonstrated efficacy in the volume-loaded rat diuresis model as well as promising in vitro and in vivo profiles and was therefore advanced to clinical development.

Pharmacodynamics of selective inhibition of γ-secretase by avagacestat.

A hallmark of Alzheimer's disease (AD) pathology is the accumulation of brain amyloid ß-peptide (Aß), generated by γ-secretase-mediated cleavage of the amyloid precursor protein (APP). Therefore, γ-secretase inhibitors (GSIs) may lower brain Aß and offer a potential new approach to treat AD. As γ-secretase also cleaves Notch proteins, GSIs can have undesirable effects due to interference with Notch signaling. Avagacestat (BMS-708163) is a GSI developed for selective inhibition of APP over Notch cleavage. Avagacestat inhibition of APP and Notch cleavage was evaluated in cell culture by measuring levels of Aß and human Notch proteins. In rats, dogs, and humans, selectivity was evaluated by measuring plasma blood concentrations in relation to effects on cerebrospinal fluid (CSF) Aß levels and Notch-related toxicities. Measurements of Notch-related toxicity included goblet cell metaplasia in the gut, marginal-zone depletion in the spleen, reductions in B cells, and changes in expression of the Notch-regulated hairy and enhancer of split homolog-1 from blood cells. In rats and dogs, acute administration of avagacestat robustly reduced CSF Aß40 and Aß42 levels similarly. Chronic administration in rats and dogs, and 28-day, single- and multiple-ascending-dose administration in healthy human subjects caused similar exposure-dependent reductions in CSF Aß40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Aß levels without causing Notch-related toxicities. Our results demonstrate the selectivity of avagacestat for APP over Notch cleavage, supporting further evaluation of avagacestat for AD therapy.

Design and characterization of a novel fluorinated magnetic resonance imaging agent for functional analysis of bile Acid transporter activity.

PURPOSE: To synthesize a trifluorinated bile acid that can be used for (19)F magnetic resonance imaging (MRI) of bile acid enterohepatic circulation, characterize its in vitro transporter affinity, stability, and (19)F-MRI signal, and assess its ability to concentrate in the gallbladder of C57BL/6 mice. METHODS: Target compound CA-lys-TFA was synthesized and tested for affinity toward the apical sodium dependent bile acid transporter (hASBT) and the Na+/taurocholate cotransporting polypeptide (hNTCP). In a pilot study, fasted mice were gavaged with vehicle control, 150 mg/kg or 300 mg/kg CA-lys-TFA. CA-lys-TFA in gallbladder, liver and plasma at t = 5 h was quantified. Additionally, a 24-h time course (24 mice across eight time points) was studied using 50 mg/kg CA-lys-TFA. RESULTS: CA-lys-TFA was a potent substrate of hASBT (Kt = 39.4 µM, normalized Vmax = 0.853) and hNTCP (Kt = 8.99 µM, normalized Vmax = 0.281). (19)F MRI phantom imaging showed linear signal-concentration dependence. In vivo studies showed that rapid accumulation of CA-lys-TFA in the gallbladder was maximal within 4-7 h. CONCLUSIONS: These findings suggest that CA-lys-TFA, a fluorinated non-radioactive bile acid analogue, has potential for use in MRI to measure in vivo bile acid transport and diagnose bile acid malabsorption and other conditions associated with impaired bile acid transport.

Mechanistic investigation of liver injury induced by BMS-932481, an experimental ɣ-secretase modulator.

BMS-932481 was designed to modulate ɣ-secretase activity to produce shorter and less amyloidogenic peptides, potentially averting liabilities associated with complete enzymatic inhibition. Although it demonstrated the intended pharmacology in the clinic, BMS-932481 unexpectedly caused drug-induced liver injury (DILI) in a multiple ascending dose study characterized by dose- and exposure-dependence, delayed onset manifestation, and a high incidence of hepatocellular damage. Retrospective studies investigating the disposition and probable mechanisms of toxicity of BMS-932481 are presented here. These included a mass balance study in bile-duct-cannulated rats and a metabolite profiling study in human hepatocytes, which together demonstrated oxidative metabolism followed by biliary elimination as the primary means of disposition. Additionally, minimal protein covalent binding in hepatocytes and lack of bioactivation products excluded reactive metabolite formation as a probable toxicological mechanism. However, BMS-932481 and 3 major oxidative metabolites were found to inhibit the bile salt export pump (BSEP) and multidrug resistance protein 4 (MRP4) in vitro. Considering human plasma concentrations, the IC50 values against these efflux transporters were clinically meaningful, particularly in the high dose cohort. Active uptake into human hepatocytes in vitro suggested the potential for hepatic levels of BMS-932481 to be elevated further above plasma concentrations, enhancing DILI risk. Conversely, measures of mitochondrial functional decline in hepatocytes treated with BMS-932481 were minimal or modest, suggesting limited contributions to DILI. Collectively, these findings suggested that repeat administration of BMS-932481 likely resulted in high hepatic concentrations of BMS-932481 and its metabolites, which disrupted bile acid transport via BSEP and MRP4, elevating serum biomarkers of liver injury.

Phenacetin pharmacokinetics in CYP1A2-deficient beagle dogs.

Phenacetin is widely used as an in vitro probe to measure CYP1A2 activity across species. To investigate whether phenacetin can be used as an in vivo probe substrate to phenotype CYP1A2 activity in dogs, beagle dogs previously genotyped for a single nucleotide polymorphism that yields an inactive CYP1A2 protein were selected and placed into one of three groups: CC (wild-type), CT (heterozygous), or TT (homozygous mutants). The dogs were dosed with phenacetin orally at 5 and 15 mg/kg and intravenously at 15 mg/kg. Plasma samples were analyzed by liquid chromatography-tandem mass spectrometry, and phenacetin and its primary metabolite, acetaminophen, were monitored. After intravenous dosing, all groups showed similar exposure of phenacetin irrespective of genotype. After oral dosing at 15 mg/kg, the exposure of phenacetin in CC and CT dogs was similar, but phenacetin exposure was 2-fold greater in TT dogs. Exposure of the metabolite, acetaminophen, was similar in all groups; however, the mean acetaminophen/phenacetin ratio in TT dogs was 1.7 times less than that observed in CC dogs. Similar trends between the groups of dogs with respect to phenacetin exposure were also observed after a lower 5 mg/kg p.o. dose of phenacetin; however, a proportionally greater amount of acetaminophen was generated. Although oral exposure of phenacetin was 2-fold higher and acetaminophen exposure was 2-fold lower in CYP1A2-deficient (TT) dogs, these results were considered modest and suggest that phenacetin is not a selective or robust in vivo probe to measure CYP1A2 enzyme activity in the dog.

MGAT2 inhibitor decreases liver fibrosis and inflammation in murine NASH models and reduces body weight in human adults with obesity.

Monoacylglycerol acyltransferase 2 (MGAT2) is an important enzyme highly expressed in the human small intestine and liver for the regulation of triglyceride absorption and homeostasis. We report that treatment with BMS-963272, a potent and selective MGAT2 inhibitor, decreased inflammation and fibrosis in CDAHFD and STAM, two murine nonalcoholic steatohepatitis (NASH) models. In high-fat-diet-treated cynomolgus monkeys, in contrast to a selective diacylglycerol acyltransferase 1 (DGAT1) inhibitor, BMS-963272 did not cause diarrhea. In a Phase 1 multiple-dose trial of healthy human adults with obesity (NCT04116632), BMS-963272 was safe and well tolerated with no treatment discontinuations due to adverse events. Consistent with the findings in rodent models, BMS-963272 elevated plasma long-chain dicarboxylic acid, indicating robust pharmacodynamic biomarker modulation; increased gut hormones GLP-1 and PYY; and decreased body weight in human subjects. These data suggest MGAT2 inhibition is a promising therapeutic opportunity for NASH, a disease with high unmet medical needs.

Monosubstituted γ-lactam and conformationally constrained 1,3-diaminopropan-2-ol transition-state isostere inhibitors of ß-secretase (BACE).

The synthesis, evaluation, and structure-activity relationships of a class of γ-lactam 1,3-diaminopropan-2-ol transition-state isostere inhibitors of BACE are discussed. Two strategies for optimizing lead compound 1a are presented. Reducing the overall size of the inhibitors resulted in the identification of γ-lactam 1i, whereas the introduction of conformational constraint on the prime-side of the inhibitor generated compounds such as the 3-hydroxypyrrolidine inhibitor 28n. The full in vivo profile of 1i in rats and 28n in Tg 2576 mice is presented.

Synthesis and SAR of indole-and 7-azaindole-1,3-dicarboxamide hydroxyethylamine inhibitors of BACE-1.

Heterocyclic replacement of the isophthalamide phenyl ring in hydroxyethylamine (HEA) BACE-1 inhibitors was explored. A variety of indole-1,3-dicarboxamide HEAs exhibited potent BACE-1 enzyme inhibition, but displayed poor cellular activity. Improvements in cellular activity and aspartic protease selectivity were observed for 7-azaindole-1,3-dicarboxamide HEAs. A methylprolinol-bearing derivative (10n) demonstrated robust reductions in rat plasma Aß levels, but did not lower rat brain Aß due to poor central exposure. The same analog exhibited a high efflux ratio in a bidirectional Caco-2 assay and was likely a substrate of the efflux transporter P-glycoprotein. X-ray crystal structures are reported for two indole HEAs in complex with BACE-1.

Synthesis and in vivo evaluation of cyclic diaminopropane BACE-1 inhibitors.

The synthesis, evaluation, and structure-activity relationships of a set of related constrained diaminopropane inhibitors of BACE-1 are described. The full in vivo profile of an optimized inhibitor in both normal and P-gp deficient mice is compared with data generated in normal rats.

Discovery of an Oxycyclohexyl Acid Lysophosphatidic Acid Receptor 1 (LPA1) Antagonist BMS-986278 for the Treatment of Pulmonary Fibrotic Diseases.

The oxycyclohexyl acid BMS-986278 (33) is a potent lysophosphatidic acid receptor 1 (LPA1) antagonist, with a human LPA1 Kb of 6.9 nM. The structure-activity relationship (SAR) studies starting from the LPA1 antagonist clinical compound BMS-986020 (1), which culminated in the discovery of 33, are discussed. The detailed in vitro and in vivo preclinical pharmacology profiles of 33, as well as its pharmacokinetics/metabolism profile, are described. On the basis of its in vivo efficacy in rodent chronic lung fibrosis models and excellent overall ADME (absorption, distribution, metabolism, excretion) properties in multiple preclinical species, 33 was advanced into clinical trials, including an ongoing Phase 2 clinical trial in patients with lung fibrosis (NCT04308681).